The present invention relates to polypeptides comprising IL-6 ligand-binding receptor domains, nucleic acids encoding such polypeptides, antibodies, compositions comprising such polypeptides, nucleic acids, or antibodies, and methods of use.
Interleukin-6 (IL-6) is a cytokine that is produced in response to various stimulators and is responsible for a variety of biological activities, including the stimulation of B- and T-cell growth and differentiation (Muraguchi et al., J. Exp. Med. 167: 332 (1988)), production of acute-phase proteins in response to inflammation or tissue injury (Gauldie et al., PNAS USA 84: 7251 (1987); Geiger et al., Eur. J. Immunol. 18: 717 (1988)), multilineage hematopoiesis, osteoclast formation, maturation of megakaryocytes, and platelet production. These biological activities are initiated when IL-6 binds to the extracellular portion of the interleukin-6 receptor, which is variously referred to as the interleukin-6xcex1 subunit (IL-6Rxcex1) or B-cell stimulating factor receptor (BSF-2 receptor). When IL-6 binds to IL-6Rxcex1, a complex is formed. The complex then binds to the extracellular portion of the interleukin-6 receptor known as gp130, which is also referred to as the interleukin-6xcex2 subunit (IL-6Rxcex2). The resulting complex then transmits the IL-6 signal intracellularly.
The precursor of the IL-6 receptor reportedly comprises 468 amino acids (Yamasaki et al., Science 241: 825-828 (1988)). The mature IL-6 receptor reportedly comprises 449 amino acids (Yamasaki et al. (1988), supra).
Abnormal expression of IL-6 has been implicated in the pathogenesis of a variety of diseases, including multiple myeloma, plasmacytoma, hematological diseases such as plasma cell dyscrasias, leukemia and lymphoma (including non-Hodgkins""s lymphoma and Lennert""s T-cell lymphoma (Kishimoto, Blood 74: 1 (1989)), mesangial proliferative glomerulonephritis, polyclonal B-cell activation conditions, allergic diseases (Type I-IV), rheumatoid arthritis (Hirano et al., Eur. J. Immunol. 18: 1797 (1988)), diabetes, multiple sclerosis, SLE, septic shock, bacterial infection, viral infection, post-menopausal osteoporosis, chronic immune deficiency and autoimmune diseases (Med. Immunol. 15: 195-201 (1988)), including organ-specific and systemic diseases and AIDS, inflammatory diseases, and Cattleman""s disease. In addition, IL-6 production has been associated with cardiac myxoma and cervical cancer (Kishimoto et al., Ann. Rev. Immunol. 6: 485 (1988)) in vivo and myelomas, histiocytomas and promyelocytic leukemia (Taga et al., J. Exp. Med. 166: 967 (1987)) in vitro. Attempts to abrogate the effects of abnormal expression of IL-6 can be made at its site of production or at its target.
In view of the above, there remains a need for materials and methods for identifying and designing agents that inhibit IL-signaling and for treating diseases involving IL-6 signaling prophylactically and therapeutically. It is an object of the present invention to provide such materials and methods. This and other objects and advantages, as well as additional inventive features, will become apparent from the detailed description provided herein.
The present invention provides, among other things, a polypeptide, and a pharmaceutically acceptable salt thereof, that inhibits the binding of IL-6 ligand with IL-6 receptor under physiological conditions. In one embodiment, the polypeptide has the formula R1R*R*L*L*L*R*R2, and pharmaceutically acceptable salts thereof, in which R1 is hydrogen, R3C(O)xe2x80x94 or R3, and does not comprise an amino acid residue sequence that is identical to an amino acid residue sequence of the xcex1-chain of the IL-6 receptor and is not linked to the moiety xe2x80x94R*R*L*L*L*R* via a glycinyl residue or via a proprionyl residue, R2 is hydrogen, a polypeptide of from 1 to about 100 amino acid residues, NHR3 or R3, and R3 is a pharmaceutically acceptable substituent group.
In another embodiment, the polypeptide has the formula R10R11XVL*2L*2VR12, in which R10 and R12, independently, are pharmaceutically acceptable substituents, R11 is a naturally-occurring or synthetic amino acid residue that has an acidic or neutral side-chain under physiological conditions, X is any naturally-occurring or synthetic amino acid residue, and L*2 is leucinyl or isoleucinyl.
In yet another embodiment, the polypeptide has the formula R20R21L*R*Y*R*A*E*R*S*R22, in which R20 and R22 are pharmaceutically acceptable substituents, R21 is a naturally-occurring or synthetic amino acid residue that has a basic or neutral side-chain under physiological conditions, L*, Y*, E* and S* are independently any naturally-occurring or synthetic amino acid residue, R* is a naturally-occurring or synthetic amino acid residue that has a basic side-chain under physiological conditions, and A* is alaninyl, glycinyl, isoleucinyl, leucinyl, valinyl, norleucinyl, norvalinyl, sarcosinyl, xcex2-alaninyl or xcex1-aminoisobutyryl.
In still yet another embodiment, the polypeptide comprises at least I*A*I*V*L*R*F* but less than about 200 amino acid residues that have a sequence that is identical to an amino acid sequence of the xcex1-chain of the IL-6 receptor, in which I*, L*, and V* are independently a naturally-occurring or synthetic amino acid residue having a side-chain consisting of a C1-C6 straight chain or C1-C6 branched alkyl moiety, R* is a naturally-occurring or synthetic amino acid residue that has a basic side-chain under physiological conditions, A* is alaninyl, glycinyl, isoleucinyl, leucinyl, valinyl, norleucinyl, norvalinyl, sarcosinyl, xcex2-alaninyl or xcex1-aminoisobutyryl, and F* is tyrosinyl, phenylalaninyl, tryptophanyl or xcex1-aminoisobutyryl, with the proviso that at least four of the seven substituents of I*A*I*V*L*R*F* are selected such that I* is isoleucinyl, A* is alaninyl, V* is valinyl, L* is leucinyl, R* is argininyl, and F* is phenylalaninyl.
In a further embodiment, the polypeptide comprises up to 200 amino acid residues that are identical to an amino acid residue sequence of the xcex2-chain of the IL-6 receptor and comprises the sequence SVIILKYNIQY (SEQ ID NO:6), TRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVL (SEQ ID NO:7), QLPVDVQNGFIRNYTIFYRTIIGN (SEQ ID NO:8), or IVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIA (SEQ ID NO:9), any one of which can comprise from one to about six conservative or neutral replacements. The polypeptide can further comprise a pharmaceutically acceptable substituent.
Also provided by the present invention is a nucleic acid that encodes an above-described polypeptide, wherein the polypeptide preferably consists of naturally-occurring amino acid residues. The nucleic acid encoding the polypeptide can be expressed in a cell. The nucleic acid encoding the polypeptide can be operably linked to a signal sequence that causes secretion of at least the polypeptide by a cell in which the nucleic acid is expressed. Alternatively, the nucleic acid comprises or encodes an antisense nucleic acid molecule or a ribozyme that is specific for a nucleotide sequence in a nucleic acid encoding the specified amino acid sequence in an above-described polypeptide.
Further provided by the present invention is a composition comprising an above-described polypeptide or nucleic acid and a carrier therefor. Another composition provided by the present invention is a composition comprising an antibody to an above-described polypeptide, an anti-antibody to an above-described polypeptide, or a solid support matrix to which is attached an above-described polypeptide or an anti-antibody to the polypeptide sequence RRLLLR (SEQ ID NO:10), RXVLLV (SEQ ID NO:11), LRYRAERS (SEQ ID NO:12), IAIVLRF (SEQ ID NO:13), SVIILKYNIQY (SEQ ID NO:6), PSIKSVIILKYNIQY (SEQ ID NO:14), or a portion of any of the following polypeptides: WTNPSIKSVIILKYNIQY (SEQ ID NO:15), KLTWTNPSIKSVIILKYNIQY (SEQ ID NO:16), TRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVL (SEQ ID NO:7), QLPVDVQNGFIRNYTIFYRTIIGN (SEQ ID NO:8), and IVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIA (SEQ ID NO:9).
Also provided by the present invention is a method of prophylactically or therapeutically inhibiting IL-6 signaling in a mammal. The method comprises administering to a mammal in need thereof an IL-6 signaling inhibiting effective amount of an above-described polypeptide, a nucleic acid encoding such a polypeptide or an antibody to such a polypeptide.
In addition, the present invention provides a method of removing IL-6 ligand from a bodily fluid of an animal. The method comprises extracorporeally contacting the bodily fluid of the animal with a solid-support matrix to which is attached an above-described polypeptide or an anti-antibody to the polypeptide sequence RRLLLR (SEQ ID NO:10), RXVLLV (SEQ ID NO:11), LRYRAERS (SEQ ID NO:12), IAIVLRF (SEQ ID NO:13), SVIILKYNIQY (SEQ ID NO:6), PSIKSVIILKYNIQY (SEQ ID NO:14), or a portion of any of the following polypeptides: WTNPSIKSVIILKYNIQY (SEQ ID NO:15), KLTWTNPSIKSVIILKYNIQY (SEQ ID NO:16), TRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVL (SEQ ID NO:7), QLPVDVQNGFIRNYTIFYRTIIGN (SEQ ID NO:8), and IVVPVCLAFLLTTLLGVLFCFNKRDLIKKHIWPNVPDPSKSHIA (SEQ ID NO:9). Alternatively, the bodily fluid can be contacted with the polypeptide or anti-antibody in solution and then the solution can be contacted with a solid support matrix to which is attached a means to remove the polypeptide or anti-antibody to which is bound IL-6 ligand from the bodily fluid.